In the field of biochemistry, it is commonly known to employ the fluorescence detecting technique for the detection of samples. Because the fluorescence detecting technique does not need to contact with to-be-test samples, non-destructive quantitative or qualitative measurements of the to-be-test samples can be done by operators without exposing to highly contagious environment. This leads to wider usage of the technique.
Fluorescence detection is to utilize certain components of an analyte being bound to a specific fluorescent dye which has a photoluminescence characteristic for a specific wavelength of light spectrum. Consequently, by illuminating the analysis of samples under a constant-intensity incident light, emission spectrums of the samples can be measured as analyzing types or concentrations of the samples. Common fluorescent dyes comprises FAM in blue-light band, HEX in green-light band, TAMRA in yellow-light band, ROX in orange-light band, CY5 in red light band, and Red670 in deep red light band.
Polymerase chain reaction (PCR) is a molecular biology technique, which can be used to expand specific DNA fragments with appropriate agents and thermal cycle equipment. And after each thermal cycle, when the reactants are detected by a fluorescence detecting system, a method for qualitative analysis or quantitative analysis for the total amount of specific products in the reactants is a real-time polymerase chain reaction (Real-time PCR).
In the field of biochemistry, multi-channel fluorescence detection typically employs real-time/quantitative polymerase chain reaction (real-time PCR or QPCR) methods. Furthermore, due to the diversity of to-be-test samples and reagents, the light source of the equipment generally comprises one or more wavelength bands of ultraviolet, visible or infrared.
Referring to FIG. 1, a schematic diagram of a fluorescence detecting system 10 of a conventional art is shown. The fluorescence detecting system 10 includes a light source 11, an excitation-light guide tube 12, a fluorescence labeled analyte 13, an emission-light guide tube 14, an emission-light filter 15, and a detector 16. The operating flow is as follows. The light source 11 emits an excitation light 17; next, the excitation light 17 enters the excitation-light guide tube 12; next, the excitation light 17 illuminates the fluorescence labeled analyte 13 to generate an emission light 18; next, the emission light 18 enters the emission-light guide tube 14; next, the emission light 18 enters the detector 16 through the emission-light filter 15; and finally, the detector 16 determines a type of the emission light 18. In general, in the present field, the excitation light 17 is a light that is used to illuminate the fluorescence labeled analyte 13, the emission light 18 is a light that has been reflected from the fluorescence labeled analyte 13. In general, the excitation-light guide tube 12 and the emission-light guide tube 14 are two paths independent from each other. This would increase the difficulty in assembly of the fluorescence detecting system 10.
In order to speed up and simplify the fluorescence detecting process, the light source 11 is capable of providing a variety of wavelength bands, and the emission-light filter 15 is a multiband emission filter.
In general, the conventional fluorescence detecting system employs a white light as a light source for rapid detection, since the fluorescence labeled analyte 13 may generates photoluminescence for a variety of wavelength bands. When the excitation light 17 illuminates the fluorescence labeled analyte 13 and further generates an emission light 18 having a plurality of wavelength bands is correspondingly generated; in other word, the emission light 18 comprises a variety of wavelength bands, correspondingly. Next, the emission light 18 is filtered by the emission-light filter 15, to produce alight with different wavelength bands. Thus, when the fluorescence labeled analyte 13 reacts for the light with different wavelength bands, it is possible to perform the detection, simultaneously, whereby the consumed time is reduced.
Although the conventional art can simultaneously detect a variety of wavelength bands, there are several existing problems therein. 1. When the light is white light, excessive emission light is generated therefrom; 2. Since when the emission light 18 passes through the emission-light filter 15, some wavelength bands, which do not need to be filtered, might be filtered out by the emission-light filter 15, thereby affecting the accuracy of the follow-up detection; 3. The emission-light filter 15 is made with employment of multi-layer filters on the same glass piece by a special way, so that its production process and production cost get higher; 4. Although white light can be employed to emit light with a variety of wavelength bands at the same time, it may cause the emission light 18 generating other lights with unnecessary wavelength bands. The results in a miscarriage of justice.
Referring to FIG. 2, a schematic diagram of a fluorescence detecting system 20 of another conventional art is shown. A difference of the fluorescence detecting system 20 from the fluorescence detecting system 10 is addition of an excitation-light filter 19 into before the excitation light 17 is illuminated onto the fluorescence labeled analyte 13. More specifically, the excitation-light filter 19 is arranged between the light source 11 and the excitation-light guide tube 12 in the another conventional art.
Although the fluorescence detecting system 20 improves the efficiency of the fluorescence detection system 10 by providing the excitation-light filter 19. However, since when the emission light 18 passes through the emission-light filter 15, the emission light 18 may be filtered in those wavelength bands which does not need to be filtered, thus affecting the accuracy of subsequent detection, such that other technical problems are still un-solved.
Furthermore, in another conventional art, a fluorescence detecting system 20 employs a plurality of sensors to rapidly perform a detection operation, but a plurality of sensors still brings the system cost increased and assembly difficult.
In summary, the conventional arts almost employ a light source (i.e., a white light) which is capable of generating multi-wavelength bands in order to increase detection efficiency but reducing the accuracy of detection for a single wavelength band.
Such as Thermo Fisher QuantStudio 12K Flex and Roche LightCycler 480 and other products, are used in the visible band of white LED fluorescence detecting system as light sources, of the white LED light, these kinds of white LED has become the mainstream of visible light source for the emitting wavelength of the white LED can cover the visible light wavelength band, sufficient brightness, cheap, light weight, small size, low power consumption, and vibration resistance.
A generation mechanism of emitting white light by the white light emitting diode is to apply a voltage on a GaN PN diode chip which is capable of emitting blue light, and to arrange a fluorescence substance, which can be excited by the blue light, in front of the blue light chip, whereby after excitated, the fluorescence substance correspondingly generates fluorescence light, such as in green, yellow, red, in order to accomplish the generation of white light. The spectral distribution of the white light emitting diodes mainly depend on a superposition among emission wavelengths of the blue light LED chip and the various fluorescent light generated by the various fluorescent substances. When the white LED power is activated, a fixed spectral distribution of visible light can be given; namely, as long as light intensity of each wavelength in the spectrum is fixed, their wavelengths and color brightness are unable to be controlled. Therefore, in real-time PCR application, if intending to strengthen the intensity of a certain color excitation light, we can only apply higher voltage on the blue LED chip, so that the overall white LED light-emitting bands become brighten at the same time, and then the light need to pass through the filter so as to derive a higher-brightness monochromatic light. This causes the overall energy consumption efficiency of such a white LED light source lowered as well as easily causing unnecessary waste of light and electricity. When a greater current passes through the LED chip, more heat is generated therein, so that the conversion efficiency from electric energy to light is low, and also aging of the light source is accelerated. In addition, for a long time, there is no fluorescent substance suitably cooperated with the white LED in the deep red band. This leads to a puzzle of applying the white LED in the red band.
To complement the shortcomings of a single white LED, some real-time PCR detector manufacturers, such as TOptical Thermocycler in Germany, Biometra, in the light source of the fluorescence detection area, besides using the white LED, respectively adds a red LED and a blue LED in the optical paths so as to compensate the issue of insufficient light-emitting band. However, this causes that the optical design becomes complicated, and the instability of the instrument is raised.
In another conventional art, a fluorescence detecting system employs four sensors to constitute a four-channel real-time PCR detector which allows scanning four fluoroscopic channels for one scan process, thereby reducing detection times. However, such a design of synchronous detection does not only increase the cost of optical sensors and optical paths, but also causes that the overall sensing area becomes large and complex.
As mentioned above, a plurality of LEDs are employed so as to compose a white light in the conventional art. However, the conventional fluorescence detecting system has to turn on all of the plurality of LEDs, in order to detect all the fluorescent labels at the same time: otherwise, the purpose of synchronous detection is unable to achieve.
Since most of the detections are done for specific objects, it is technical problems, which are urgent to be solved, of how to effectively increase the accuracy of detection of a specific wavelength band, effectively increase the luminous flux of a specific wavelength band (without increasing the full wavelength of the flux under the premise), and simplify the structure of the fluorescence detecting system and reduce production costs.
Hence, it is essential to provide a multi-channel fluorescence detecting system and a method of using the same to solve the above technical problems.